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Functional and antigenic domains of the matrix (M1) protein of influenza A virus
Abstract:
The membrane- and ribonucleocapsid (RNP)-binding domains of the matrix (M1) protein of influenza A virus (WSN strain) were partially mapped and characterized by reactivity with monoclonal antibodies (MAb) as well as by proteolytic cleavages and amino acid sequencing of the resulting peptides. Of two peptides formed by formic acid hydrolysis, a 9-kilodalton fragment at the amino-terminal third of the M1 protein was recognized by MAb M2-1C6 (to epitope 1), and a 15-kilodalton fragment at the carboxy-terminal two-thirds was recognized by MAb 289/4 (to epitope 2). Partial cleavage by staphylococcal V8 protease gave rise to a 16-kilodalton peptide, mapping to amino acid 8, which was recognized by MAbs to all three epitopes but rather weakly by MAb 904/6 to epitope 3. These studies suggest that epitope 1 of the M1 protein resides between amino acids 8 and 89, whereas epitopes 2 and possibly 3 are located between amino acids 89 and 141 or somewhat more carboxy distal. The intact M1 protein and its N-terminal 9- and 10-kilodalton peptides generated by formic acid or V8 protease cleavage, respectively, reconstituted with dipalmitoylphosphatidylcholine vesicles, but these N-terminal peptides had little effect on in vitro transcription of the RNP core. In sharp contrast, both intact M1 protein and the C-terminal 15-kilodalton formic acid fragment were able to inhibit viral transcription markedly. Moreover, MAb 289/4 (to epitope 2) reversed this inhibited transcription significantly. These studies suggest that the lipid-binding domain of the M1 protein is located within the amino-terminal third, whereas the site involved in the interaction of the M1 protein with RNP cores is located within the carboxy-terminal two-thirds.
Insights
The influenza A virus matrix protein (M1) has distinct domains for binding membranes and ribonucleocapsids (RNPs). The M1 protein
Area of Science:
- Virology
- Molecular Biology
- Protein Chemistry
Background:
- The matrix (M1) protein of influenza A virus is crucial for viral assembly and budding.
- Understanding the M1 protein's domains is essential for comprehending viral replication mechanisms.
Purpose of the Study:
- To map and characterize the membrane- and ribonucleocapsid (RNP)-binding domains of the influenza A virus M1 protein.
- To investigate the functional roles of different M1 protein fragments in viral transcription and membrane interaction.
Main Methods:
- Utilized monoclonal antibodies (MAbs) to map epitopes on the M1 protein.
- Employed proteolytic cleavages (formic acid, staphylococcal V8 protease) and amino acid sequencing to generate and analyze M1 protein fragments.
- Assessed the ability of M1 protein fragments to reconstitute with lipid vesicles and affect in vitro viral transcription.
Main Results:
- Identified distinct regions within the M1 protein responsible for lipid binding (amino-terminal third) and RNP interaction (carboxy-terminal two-thirds).
- Epitope 1 is located between amino acids 8 and 89; epitopes 2 and potentially 3 are between amino acids 89 and 141 or further C-terminally.
- N-terminal M1 fragments reconstituted with lipid vesicles but had minimal impact on RNP transcription, while C-terminal fragments significantly inhibited transcription.
Conclusions:
- The M1 protein possesses separate domains for membrane association and RNP binding.
- The carboxy-terminal domain of M1 protein plays a critical role in regulating viral transcription through RNP interaction.
- Monoclonal antibody 289/4, targeting epitope 2, can reverse M1-mediated inhibition of viral transcription.